A comparison of ELISA and RPLA for detection of Bacillus cereus diarrhoeal enterotoxin

Fourteen strains of Bacillus cereus isolated from different sources were examined for their ability to produce diarrhoeal enterotoxin by two commercial immunoassay kits (Oxoid BCET-RPLA and Tecra ELISA) and the microslide immunodiffusion assay. One strain that was positive in monkey feedings, as well as a number of other strains isolated from diarrhoeal outbreaks, gave positive results in the ELISA and negative results in the RPLA test systems. When tested in the microslide assay, these strains produced only one antigen which formed a line of identity with the reference toxin. The results of the control toxins provided with the kits substantiated that the two commercial assays did not detect the same antigen. Cultures positive with both assay kits were shown to produce diarrhoeal enterotoxin (by a line of identity) and other antigens in the microslide immunodiffusion assay.     

Improved cytotoxicity assay for Bacillus cereus diarrhoeal enterotoxin

An improved McCoy cell cytotoxicity assay for Bacillus cereus diarrhoeal toxin, which includes a staining procedure in addition to visual examination, was developed and the method was compared with two commercially available kits (Oxoid BCET-RPLA and Tecra BDE-VIA). A total of 71 strains of 15 different Bacillus, Brevibacillus and Paenibacillus species, including 16 strains of B. cereus from outbreaks of food-borne illness, were evaluated for toxin production. Eleven of the outbreak strains exhibited cytotoxicity, including all six B. cereus strains associated with diarrhoeal illness. Several other isolates of B. cereus, and its relatives B. anthracis, B. mycoides and B. thuringiensis, exhibited similar cytotoxicity. The other species showed no cytotoxicity, with the exception of certain B. subtilis strains. The cytotoxicity assay was more sensitive than the Oxoid kit and unlike the Tecra kit, did not give false positive results with supernatant fluids heat-treated to destroy the toxin.     

Diarrhoeal enterotoxin production by strains of Bacillus thuringiensis isolated from commercial Bacillus thuringiensis-based insecticides

Abstract Strains of Bacillus cereus and B. thuringiensis were tested by the Tecra VIA kit for the ability to produce a diarrhoeal enterotoxin. The strains of B. thuringiensis were isolated from commercial B. thuringiensis -based insecticides (Bactimos^TM, DiPel^TM, Florbac^TM FC, Foray^TM 48B, Novodor^TM FC, Turex^TM, VecTobac^TM, XenTari^TM). The production of diarrhoeal enterotoxin varied by a factor of more than 100 among the different strains tested. B. cereus (F4433/73) produced the highest amount of enterotoxin and the B. thuringiensis strain isolated from DiPel^TM the lowest. The products were tested for their content of diarrhoeal enterotoxin and all products, except MVP^TM which does not contain viable B. thuringiensis spores, contained diarrhoeal enterotoxins. The results indicates an potential risk for gastroenteritis outbreak caused by B. thuringiensis .     

Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks

Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food-poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET-RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.     

Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay

An improved qualitative cell cytotoxicity assay for the detection of Bacillus cereus emetic and enterotoxin is described. The presence of toxin in culture supernatant fluids was detected by measurement with the tetrazolium salt MTT, as it adversely affects the metabolic status of cultured CHO cells. Psychrotrophic B. cereus isolates (65) were assessed for toxin production using the cytotoxicity assay, and 91% of culture supernatant fluids were cytotoxic. Toxin assessment using BCET-RPLA and ELISA immunoassays indicated that 51% and 85% of the cultures, respectively, were toxigenic. There were pronounced strain differences in the amount of toxin produced by the B. cereus isolates. Some isolates of B. circulans, B. laterosporus/cereus, B. lentus, B. licheniformis, B. mycoides, B. subtilis and B. thuringiensis were also toxigenic.     

苏云金芽胞杆菌肠毒素基因的PCR检测

采用多重引物PCR进行了 45株苏云金芽胞杆菌、2株蜡状芽胞杆菌和 2株球形芽胞杆菌溶血素BL ,肠毒素T和entS基因的检测 ,结果表明 95 6%苏云金芽胞杆菌含溶血素hblA基因 ,91 1 %含bceT基因 ,93 3%含entS基因。用两种商业化肠毒素检测试剂盒TECRA和RPLA进行所有菌株肠毒素的体外免疫测定 ,大部分苏云金芽胞杆菌和阳性蜡状芽胞杆菌都能产生不同水平的肠毒素活性 ,同hblA基因PCR检测结果基本相符。尽管DBT0 0 7和T2 4 0 0 1含有hblA基因 ,但用TECRA却检测不到肠毒素 ;Dmu39菌株不含肠毒素基因 ,但用TECRA却检测出高的肠毒素活性。苏云金芽胞杆菌BDT2 4 8和球性芽孢杆菌不含肠毒素基因和肠毒素。结果表明昆虫病原菌苏云金芽胞杆菌的安全性有待进一步研究     

The use of Bacillus diarrhoeal enterotoxin (BDE) detection using an ELISA technique in the confirmation of the aetiology of Bacillus-mediated diarrhoea

A commercially available ELISA kit was used for the detection of Bacillus diarrhoeal enterotoxin (BDE) in a variety of foods and faeces. The ability of isolates of Bacillus spp., including Bacillus cereus , to produce BDE in Brain Heart Infusion broth containing 0·1% glucose was also checked by use of the kit. Results show that 29 out of 31 B. cereus isolates were enterotoxigenic. Foods positive for preformed BDE were always contaminated with >10⁵ B. cereus cfu g⁻¹, but not all foods contaminated with large numbers of B. cereus were positive for BDE. Bacillus spp., other than one isolate which closely resembled B. subtilis , were negative for BDE production. Criteria for the confirmation of Bacillus -mediated diarrhoea should now include reports of symptoms and incubation periods consistent with the diarrhoeal form of food-poisoning by Bacillus spp., together with the results of tests for enterotoxigenicity of the Bacillus isolate, and detection of BDE in either the food and/or faeces.     

Identification and analysis of the antigens detected by two commercial Bacillus cereus diarrheal enterotoxin immunoassay kits.

The usefulness of two commercial immunoassays for the detection of diarrheal enterotoxin of Bacillus cereus is unclear because the identity of the enterotoxin(s) has not been proven and the kits detect different proteins. We found that the Bacillus cereus Enterotoxin-Reversed Passive Latex Agglutination kit (Oxoid) detects the L2 component from hemolysin BL, and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (Tecra) detects two apparently nontoxic proteins.     

反向间接胶乳凝集试验法检测粪便A型产气荚膜梭菌肠毒素的结果及评价

本文以１０种５２株供试菌分别与７个不同年龄组的健康人粪便混合,共配成３６４份模拟标本,采用反向间接胶乳凝集（ＲＰＬＡ）试验法与生物学试验法（小鼠致死试验、豚鼠皮肤血管透性因子试验,Ｖｅｒｏ细胞毒性试验）检测各标本中的Ａ型产气荚膜梭菌肠毒素（简称Ｃｐ－Ｅｎｔ）。除产气荚膜梭菌之外的其他菌种培养液２３８份标本（３４株）,ＲＰＬＡ与生物学试验结果完全一致,均为阴性。产气荚膜梭菌１２６份标本（１８株）中有７０份标本的ＲＰＬＡ同生物学诸法完全一致地检出了Ｃｐ－Ｅｎｔ．有１株７份标本（ＣｐＮＣＴＣ８７９７）的ＲＰＬＡ为阳性,而各生物学试验却均为阴性,该菌株经ＰＣＲ检查证明确为肠毒素原性产气荚膜梭菌,表明ＲＰＬＡ比生物学试验法更灵敏。有５株（ＣｐＮＣＴｃ８２３８,ＣｐＮＣＴＣ１０６１１,ＣｐＮＣＴＣ１０６１４,ＣｐＮＣＴＣ１０６１２,ＣｐＬ－５２）３５份标本ＲＰＬＡ与各生物学试验结果均为阴性,但经ＰＣＲ检吉证明该５株菌均为肠毒素原性产气荚膜梭菌,后经超声波破碎菌体提取物对其中部分菌株进行试验的结果仍然显示了ＲＰＬＡ与生物学法的一致性。有２株（ＣｐＮＣＴＣ８６８６,ＣｐＮＣＴＣ８４４９）１４份标本的所有结果均为阴性,ＰＣＲ     

Enterotoxigenic porcine and human isolates ofYersinia enterocolitica in Sweden

The production of heat-stable and heat-labile enterotoxins byYersinia enterocolitica was studied in 69 strains from healthy swine and in 24 strains from humans with acute diarrhea. All of the human strains were of serotype O3, and 20 (83%) of them produced heat-stable enterotoxin detectable in the infant mouse assay. All were negative in the Chinese Hamster Ovary (CHO) cell test for detection of heat-labile enterotoxin. Of the 69 porcine strains, which were of twelve serotypes plus 9 nontypable strains, 26 (38%) gave a positive infant mouse test. Of the porcine isolates of serotype O3, 42% were enterotoxigenic. A high incidence of enterotoxigenicity was also apparent among six other serotypes (53%). All porcine strains were negative in the CHO cell test. However, of seven culture supernatants from these porcine strains, three gave positive reactions in rabbit skin permeability tests, two of which were also positive in rabbit loop tests. Heat treatment of the supernatants abolished the reactivity in both tests. It is concluded that production of a heatstable enterotoxin is fairly common in porcine and human strains ofY. enterocolitica of serotype O3 in Sweden.     

Fate and effect of ingested Bacillus cereus spores and vegetative cells in the intestinal tract of human-flora-associated rats

The fate and effect of Bacillus cereus F4433/73R in the intestine of human-flora-associated rats was studied using bacteriological culturing techniques and PCR-denaturing gradient gel electrophoresis in combination with cell assays and immunoassays for detection of enterotoxins. In faecal samples from animals receiving vegetative cells, only few B. cereus cells were detected. Spores survived the gastric barrier well, and were in some cases detected up to 2 weeks after ingestion. Selective growing revealed no major changes in the intestinal flora during passage of B. cereus. However, denaturing gradient gel electrophoresis analysis with universal 16S rRNA gene primers revealed significant changes in the intestinal microbiota of animals dosed with spores. Vero cell assays and a commercial kit (BCET-RPLA) did not reveal any enterotoxin production from B. cereus F4433/73R in the intestinal tract.     

Diarrhoeal enterotoxin production by psychrotrophic Bacillus cereus present in reconstituted milk-based infant formulae (MIF)

One hundred reconstituted milk-based infant formulae (MIF) representative of 10 leading brands available in many European Economic Community countries were examined for psychrotrophic Bacillus cereus and for the presence of diarrhoeal enterotoxin. Of the 38 B. cereus isolates recovered from MIF, one, four and 16 strains grew at 4, 6 and 8 °C after 15 d. One (2·6%), two (5·3%) and six (15·8%) of the isolates were identified as potential psychrotrophic food poisoning strains as they were both enterotoxigenic and exhibited good growth at 4, 6 and 8 °C, respectively. Enterotoxin was not detected in MIF in which less than 5·36 log₁₀ cfu of B. cereus ml⁻¹ had grown. While psychrotrophic enterotoxigenic B. cereus strains occur occasionally in MIF, brief storage of reconstituted MIF at the recommended refrigeration temperature of 4 °C will allow this product to remain safe for consumption.     

Comparative Studies of Bacillus Cereus Strains Isolated From Various Foods and Food Poisoning Outbreaks

Certain properties of 22 Bacillus cereus strains isolated from different foods and food poisoning episodes were investigated in order to evaluate possible différences between strains isolated from diarrhoeal and vomiting type food poisoning outbreaks. None of the strains isolated from vomiting type episodes produced acid from salicin and mannose, whereas 80 and 40 % of the strains from diarrhoeal type outbreaks were positive, respectively. No association between the antibiotic sensitivity pattern or the fatty acid composition and the source of a strain could be found, although some strains differed from the general pattern of B. cereus in some instances. No significant differences in the production of the skin factor between strains isolated from the two types of outbreaks were found either. The findings of this study support the observation that the food environment itself essentially affects the enterotoxin formation of B. cereus.     

Toxin gene profiling of enterotoxic and emetic Bacillus cereus

Very different toxins are responsible for the two types of gastrointestinal diseases caused by Bacillus cereus: the diarrhoeal syndrome is linked to nonhemolytic enterotoxin NHE, hemolytic enterotoxin HBL, and cytotoxin K, whereas emesis is caused by the action of the depsipeptide toxin cereulide. The recently identified cereulide synthetase genes permitted development of a molecular assay that targets all toxins known to be involved in food poisoning in a single reaction, using only four different sets of primers. The enterotoxin genes of 49 strains, belonging to different phylogenetic branches of the B. cereus group, were partially sequenced to encompass the molecular diversity of these genes. The sequence alignments illustrated the high molecular polymorphism of B. cereus enterotoxin genes, which is necessary to consider when establishing PCR systems. Primers directed towards the enterotoxin complex genes were located in different CDSs of the corresponding operons to target two toxin genes with one single set of primers. The specificity of the assay was assessed using a panel of B. cereus strains with known toxin profiles and was successfully applied to characterize strains from food and clinical diagnostic labs as well as for the toxin gene profiling of B. cereus isolated from silo tank populations.     

Heat-Stable Enterotoxin Produced by Yersinia enterocolitica Isolated from Patients

Twenty-three strains of Yersinia enterocolitica were isolated from children with gastrointestinal illness and examined for the production of enterotoxins by using both suckling mouse and Chinese hamster ovary (CHO) cell assay systems. Six strains were found to be enterotoxigenic in the suckling mouse assay, but all strains were negative in the CHO cell assay. Enterotoxin was detected in the culture supernatant when organisms were grown at 25 C but not at 37 C. Enterotoxin in a 15-fold concentrated culture supernatant was precipitated by adding absolute ethanol to a concentration of 90%. However, after being dialyzed against distilled water in Spectra/por 6 membrane tubing, it was soluble in 80% acetone. One unit dose of partially purified enterotoxin was 5.0 μg of protein/mouse in the suckling mouse assay. The molecular weight of enterotoxin was between 10,000 and 50,000 daltons as determined by ultrafiltration. It was stable to heat (121 C × 20 min or 100 C × 60 min). These observations indicate that Y. enterocolitica isolated in Japan also produce an enterotoxin similar to the heat-stable enterotoxin of Escherichia coli. However its physicochemical properties seem to be different from those of E. coli.     

Comparison of a competitive enzyme immunoassay kit and the infant mouse assay for detecting Escherichia coli heat-stable enterotoxin

A commercial competitive enzyme immunoassay kit, Escherichia coli ST EIA, was compared with the conventional infant mouse assay for sensitivity and specificity in detecting E. coli heat-stable enterotoxin. Thirty-one of 46 strains of E. coli tested were positive by both assays, while 15 strains were negative. The sensitivity of the ST EIA kit was up to 64-fold lower than the infant mouse assay.     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.     

Rapid discrimination of cytK-1 and cytK-2 genes in Bacillus cereus strains by a novel duplex PCR system

Bacillus cereus is the causative agent of gastrointestinal diarrhoea. At least three known enterotoxins may be involved in this syndrome: nonhaemolytic (Nhe) enterotoxin, Hbl enterotoxin and cytotoxin K. Two different forms were recently described for cytotoxin K, encoded by cytK-1 and cytK-2 genes. The CytK-1 toxin appeared to carry a high toxicity, but there is currently no method available to rapidly detect and discriminate the B. cereus strains able to produce this CytK-1 form. In this study, a duplex PCR assay was developed and validated on 162 known cytotoxin-containing strains. This PCR method is the first molecular tool to provide rapid detection and discrimination of cytK-1- and cytK-2-carrying B. cereus strains.     

An improved method for detecting cytostatic toxin (emetic toxin) of Bacillus cereus and its application to food samples

Abstract We developed an improved HEp-2 cell assay method for the detection of Bacillus cereus toxin, which affects the proliferation of HEp-2 cells. The cytostatic toxin was stable upon exposure to heat, pH 2, pH 11 and trypsin, which suggests it is an emetic. Using the HEp-2 cell assay, we examined the distribution and contamination of B. cereus strains that produced an emetic toxin in various foods. Although there were 228 enterotoxin producers among 310 B. cereus strains obtained from foods, 16 of them produced the cytostatic type (emetic toxin). All of the strains that produced the cytostatic toxin were of the H.1 serotype.